VECTORS FOR TRANSCRIPTION FACTOR CLONING AND TARGET SITE IDENTIFICATION BY MEANS OF GENETIC SELECTION IN YEAST

We describe the construction of a number of vectors that can be used i n yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target si tes recognized by transcription factors. For transcription factor clon ing we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-ess ential yeast PDC6 locus. This set of plasmids was tested in a one-hybr id assay with the rice transcription factor Oshoxl, a member of the cl ass of homeodomain leucine zipper proteins. A hybrid protein of Oshoxl and the Gal4 transcriptional activation domain was shown to specifica lly activate a reporter gene construct with upstream Oshoxl binding si tes, which had been integrated at the PDC6 locus using the described v ector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomi c or random DNA fragments upstream of a reporter gene. We have constru cted two variants of a bacteriophage lambda Vector which facilitates t he construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli s huttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhoxl as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector. (C) 1998 John Wiley & Sons, Ltd.